P339: BOOSTING THE EFFICACY OF CD20-TARGETING IMMUNOTHERAPY IN B CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: M. Poprzeczko, K. Domka, A. Pastorczak, K. Fidyt, L. Komorowski, M. Winiarska, A. Dabkowska, K. Siudakowska, A. Wojciechowska, E. Patkowska, M. Firczuk
Journal: HemaSphere (2022)

Background: CD20 is a B cell-specific surface protein that appears at the stage of pre-B cells during B cell development. Currently, monoclonal antibodies (mAb) against CD20 are commonly used for immunotherapy of mature B-cell malignancies. B cell precursor acute lymphoblastic leukemia (BCP-ALL) originates from immature B cells in a bone marrow. Overall, the effects of treatment with high-dose multi-agent chemotherapy are good. However, for relapsed/refractory patients, novel treatment options (mainly immunotherapy-based) targeting B cell-specific antigens are already used in clinics and further optimized in preclinical models. Recent clinical trials conducted in Philadelphia (Ph) chromosome-negative BCP-ALL patients with CD20 expression revealed that the outcome of young adults may be improved by a combination of chemotherapy with rituximab, the anti-CD20 mAb. However, in most patients, the expression of CD20 on BCP-ALL blasts is heterogeneous and may be insufficient for effective treatment with CD20 mAb. Also, the regulation of CD20 in BCP-ALL is not well defined. Aims: The aim of this study is to identify drugs that are able to upregulate CD20 in BCP-ALL, in order to improve the efficacy of anti-CD20 mAbs in preclinical models of the disease. Methods: We tested the impact of 38 anti-cancer drugs used for the treatment of BCP-ALL as well as tested in clinical trials on the CD20 surface levels after 48hrs of incubation. For the in vitro studies, we employed cell lines and primary cells representing selected high-risk subtypes (Ph-positive, Ph-like, B-other). The levels of CD20 were determined by flow cytometry. Next, the potential of selected drugs to activate the anti-CD20 mAb, rituximab, was tested in a functional assay, Antibody-Dependent Cellular Cytotoxicity (ADCC) with Human Peripheral Blood Mononuclear Cells (PBMC) or primary NK cells, isolated from healthy donors. Results: Based on the initial screening of cell lines, we selected 10 agents which increased the level of CD20: three histone deacetylases inhibitors, one inhibitor of MDM2 protein (p53 signaling pathway), one apoptosis inducer, three kinase inhibitors, one mitosis inhibitor, and one antimetabolic agent. Importantly, we found that many tyrosine kinases inhibitors and some retinoids significantly decreased CD20 levels in BCP-ALL cell lines. Some of these findings were also confirmed in primograft BCP-ALL blasts co-cultured with OP9 murine stromal cells. Furthemore, we found that five out of ten drugs enhance antibody-dependent cytotoxicity of rituximab, in functional assay. Moreover, two of the tested drugs increased phagocytosis of primograft BCP-ALL cells by primary macrophages differentiated from human peripheral blood monocytes. Summary/Conclusion: In summary, we identified agents upregulating CD20 levels in BCP-ALL cells in vitro. More importantly, some of the selected drugs improved RTX-mediated effector mechanisms such as ADCC and immunophagocytosis. Further studies aiming to elucidate the mechanisms of CD20 upregulation and to validate our in vitro findings in murine models are ongoing.